EP2079824A2 - Modified alkoxyglycerols - Google Patents

Modified alkoxyglycerols

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Publication number
EP2079824A2
EP2079824A2 EP07819930A EP07819930A EP2079824A2 EP 2079824 A2 EP2079824 A2 EP 2079824A2 EP 07819930 A EP07819930 A EP 07819930A EP 07819930 A EP07819930 A EP 07819930A EP 2079824 A2 EP2079824 A2 EP 2079824A2
Authority
EP
European Patent Office
Prior art keywords
alkoxyglycerols
modified
acid
saturated
acyl group
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP07819930A
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German (de)
French (fr)
Other versions
EP2079824B1 (en
Inventor
Guillermo Reglero Rada
Luis Vazquez Frutos
Carlos Torres Olivares
Tiziana Fornari Reale
Francisco Javier SEÑORANS RODRIGUEZ
Fernando Moreno Egea
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidad Autonoma de Madrid
Soluciones Extractivas Alimentarias SL
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Universidad Autonoma de Madrid
Soluciones Extractivas Alimentarias SL
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Publication of EP2079824A2 publication Critical patent/EP2079824A2/en
Application granted granted Critical
Publication of EP2079824B1 publication Critical patent/EP2079824B1/en
Not-in-force legal-status Critical Current
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/003—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fatty acids with alcohols
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/075—Ethers or acetals
    • A61K31/08—Ethers or acetals acyclic, e.g. paraformaldehyde
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/25—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids with polyoxyalkylated alcohols, e.g. esters of polyethylene glycol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/03—Preparation of carboxylic acid esters by reacting an ester group with a hydroxy group
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/52—Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/587—Monocarboxylic acid esters having at least two carbon-to-carbon double bonds
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/10—Production of fats or fatty oils from raw materials by extracting
    • C11B1/104—Production of fats or fatty oils from raw materials by extracting using super critical gases or vapours
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C1/00—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids
    • C11C1/02—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils
    • C11C1/025—Preparation of fatty acids from fats, fatty oils, or waxes; Refining the fatty acids from fats or fatty oils by saponification and release of fatty acids
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/08—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils with fatty acids
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom
    • C11C3/04—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom by esterification of fats or fatty oils
    • C11C3/10—Ester interchange
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6454—Glycerides by esterification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6472—Glycerides containing polyunsaturated fatty acid [PUFA] residues, i.e. having two or more double bonds in their backbone
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/54—Improvements relating to the production of bulk chemicals using solvents, e.g. supercritical solvents or ionic liquids

Definitions

  • This invention relates to lipid compounds with biological activity and more specifically to lipid compounds derived from ether lipids (or alkoxyglycerols) with a beneficial activity for health, or which can be lipid carriers for the administration of bioactive ingredients.
  • the invention further relates to the process for obtaining said modified alkoxyglycerols.
  • Shark liver oil is a natural source of certain alkoxyglycerols and other types of lipids such as triacylglycerols, squalene and cholesterol.
  • Natural alkoxyglycerols or 1-O-alkyl-sn-glycerols are valuable compounds which can be isolated from shark liver oil. Different applications of such compounds naturally present in fish oils, and particularly in shark liver oils, have been described, such as the use of these 1 -O-alkyl-glycerols for preserving or improving sperm in pharmaceutical or veterinary uses (ES 2221057, W9800120).
  • a 1 -O-alkyl-glycerol compound chosen from the group comprising 16:0 1 -alkyl-glycerol, 18:1 1 -alkyl-glycerol and 16.1 1 - alkylglycerol, which are ether lipids present in shark liver oil, is used.
  • Natural ether lipids also known as alkoxyglycerols or alkyl glycerol ethers
  • the oral administration of ether lipids reduces the negative effects of radiations received in radiation therapies.
  • Prophylactic effects in certain tumors have also been observed. It has also been observed in experiments in both animals and humans that they are able to improve the immune response (Andreesen R., "Ether lipids in the therapy of cancer", Prog Biochem
  • ether lipids are found in bone marrow, breast milk, placenta and in shark liver oils in large amounts. In the human body, the ether lipids are mainly found in immune system cells and in breast milk. In some circumstances, the endogenous synthesis is reduced and in these cases, the administration of alkoxyglycerols is recommended.
  • fats and oils enriched with long chain polyunsaturated fatty acids and/or with conjugated linoleic acid they can be enzymatically enriched.
  • CLA conjugated linoleic acid
  • CLA conjugated linoleic acid
  • Nutritional studies based on animal models have demonstrated a wide variety of beneficial effects derived from ingesting CLA in the diet. These effects include their anticarcinogenic, antiatherogenic, antiobesity properties and their immune system enhancing effect (Whigham, L. D., M. E. Cook, R. L. Atkinson, "Conjugated Linoleic Acid. Implications for Human Health", Pharmacol Res. 42: 503-510, (2000), Pariza, M.W., Y. Park, M. E. Cook, "The Biologically Active Isomers of Conjugated Linoleic Acid", Prog. Lipid Res. 40: 283-298, (2001 )).
  • omega-3 fatty acids especially long-chain omega-3 fatty acids, i.e. DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), have effects on the maintenance of arterial elasticity and, accordingly, on the maintenance of normal blood pressure levels and the reduction of cardiovascular risk.
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • the researchers then determined elasticity of the arteries of the participants through ultrasounds. Those who received co-3 fatty acids showed a significant reduction in arterial sclerosis, whereas those who took placebo experienced no changes. Those who took EPA showed a 36% increase in arterial systemic resistance, a measure of elasticity of the main arteries, whereas those who took DHA showed a 27% increase.
  • Omega-3 fatty acids improve the blood lipid profile because they increase elasticity, decrease LDL cholesterol, increase HDL cholesterol, reduce arterial triglycerides and are antithrombotic.
  • Adeemia (L ⁇ pez-Huertas-E; Baro, L.; Carrero, J J; Fonolla, J (2003) "n-3 fatty acids: health effects and opportunities to increase intake", Agro Food Industry hi tech 2003, 14(3).
  • omega-3 stands out due to its involvement in forming cell membranes. Most brain tissues are rich in omega-3 fatty acids. The current knowledge of these effects is included in an article by Donald B. Jump of the Department of Physiology, Biochemistry, and Molecular Biology of Michigan State University published in the Journal of Biological Chemistry of the American Society for Biochemistry and Molecular Biology (J Biol Chem 227 (2002) 8755-8758).
  • Ether lipids can furthermore be suitable as lipid carriers to assure higher bioavailability and a better response of these substances.
  • the combined administration of both, fatty acids and alkoxyglycerols, in a single molecule enhances the beneficial effect that both molecules have separately.
  • a first object of the invention is represented by modified alkoxyglycerols of general formula CH2OR1-CHOR2-CH2OR3 where Ri, R2 and R 3 independently represent a hydrogen; a saturated or unsaturated, linear or branched C2-C2 4 alkyl group, with or without double bonds, or a conjugated or unconjugated, saturated or unsaturated, linear or branched d- C2 4 acyl group with or without double bonds where at least one of Ri, R2 or
  • R3 represents an alkyl group.
  • a second object is the process for obtaining said modified alkoxyglycerols comprising three steps: supercritical fractionation of the natural alkoxyglycerols, saponification and re-esterification thereof.
  • modified alkoxyglycerols of the invention to prevent cardiovascular diseases, as an immunostimulant or as an anticarcinogen.
  • Modified alkoxyglycerols are also useful as carrier agents for other active ingredients and bioactive substances, making them suitable to form part of food compositions in functional foods or as an additive in pharmaceutical compositions.
  • a first aspect of the present invention relates to modified alkoxyglycerols of general formula CH2OR1-CHOR2-CH2OR3 where Ri, R2 and R 3 independently represent a hydrogen; a saturated or unsaturated, linear or branched C2-C2 4 alkyl group, with or without double bonds; or a conjugated or unconjugated, saturated or unsaturated, linear or branched d-
  • the modified alkoxyglycerols Ri is a saturated or unsaturated, linear or branched C2-C2 4 alkyl; R 2 is a hydroxyl group or an alkyl or acyl group, both being C 2 -C 24 and
  • R 3 is a hydroxyl group or an alkyl or acyl group, both being conjugated or unconjugated, saturated or unsaturated, linear or branched C 2 -C 24 with or without double bonds where at least one of R 2 or R 3 represents an acyl group.
  • the acyl group or groups come from saturated fatty acids with 1 -10 carbons.
  • the acyl groups have one or more unsaturations. It is also preferred that these unsaturations are conjugated as, for example, in the case of conjugated linoleic acid (CLA) which, as an acyl substituent in the general formula, would be in the form of conjugated linoleyl radical.
  • CLA conjugated linoleic acid
  • DHA docosahexaenoic acid
  • EPA eicosapentaenoic acid
  • acyl group or groups in the modified alkoxyglycerols of the invention come from oleic acid, therefore representing an oleyl radical.
  • R1 , R2 or R3 represent an alkyl group it is preferred that they have an even number of carbons.
  • the modified alkoxyglycerols of the invention are useful for preventing cardiovascular diseases and are also useful as stimulants of the immune system and anticarcinogenic. This activity comes from the beneficial effect provided by the fatty acids, whereby modifying the alkoxyglycerols, and by the ether lipids themselves.
  • the modified alkoxyglycerols of the invention can be used in both the food and pharmaceutical industry for preparing functional products and drugs.
  • the alkoxyglycerols of the invention can be used as carrier agents of other bioactive compounds in food or pharmaceutical compositions.
  • the modified alkoxyglycerols of the invention can act as carrier agents of fatty acids of the omega-3, omega-6, omega-9 (oleic) and/or CLA family. A synergistic activity can thus be reached between the alkoxyglycerols of the invention and fatty acids carried by them.
  • Another aspect of the invention is the process for obtaining the modified alkoxyglycerols of the invention.
  • This process comprises: a) countercurrent column supercritical fractionation of shark liver oil for obtaining a refined product rich in alkoxyglycerols and an extract rich in squalene, b) saponification of the refined product rich in alkoxyglycerols of a) for producing an unsaponifiable fraction rich in non-esterified alkoxyglycerols, and c) chemical or enzymatic re-esterification of the product of b) with aliphatic, saturated or unsaturated, conjugated or unconjugated, linear or branched chain C2-C2 4 monocarboxylic acids.
  • the shark liver oil can be subjected to another optional supercritical extraction step to extract squalene and other lipid products present in the oil, such as cholesterol or triacylglycerols, to obtain a refined product rich in even purer alkoxyglycerols.
  • squalene and other lipid products present in the oil such as cholesterol or triacylglycerols
  • Supercritical fractionation is carried out in a filled column to separate ether lipid components from edible oil samples containing them.
  • Shark liver oil is particularly preferred due to its high content in alkoxyglycerols.
  • the extraction is carried out with supercritical fluids, preferably carbon dioxide, on the alkoxyglycerols source (shark liver oil, or from a shark oil chemically or enzymatically modified or modified by saponification).
  • the fractionation is carried out at an extraction pressure of between 90 and 320 bar, preferably 9-270 bar, and at a temperature of between 1 O 0 C and 9O 0 C.
  • a refined product rich in alkoxyglycerols (less than 4% of squalene) and an extract rich in squalene are obtained after fractionation.
  • Saponification process The refined product rich in alkoxyglycerols obtained after supercritical fractionation is still unsuitable for esterification because it still has a high percentage of thacylglycerols that can cause unwanted esterification and transestehfication reactions in the presence of lipases. Therefore, saponification is carried out before this step to eliminate all the triacylglycerols and to obtain an unsaponifiable fraction highly rich in non- estehfied alkoxyglycerols.
  • Saponification is carried out by means of treating the refined product rich in alkoxyglycerols with soda or potash or with an alkali in a basic medium. An unsaponifiable residue is obtained from this reaction, which product will be used in the transesterification step.
  • Esterification of the alkoxyglycerols is preferably carried out enzymatically with a lipase in the presence of C2-C2 4 fatty acids or with esters thereof, with which the alkoxyglycerols are to be estehfied.
  • the reaction catalyzed by the lipase takes place in two consecutive steps.
  • An alkylglycerol monoester is formed during the first acylation and the alkylglycerol diester is formed during a second acylation.
  • modified alkoxyglycerols, different from natural alkoxyglycerols are thus obtained.
  • Conjugated linoleic acid (CLA), eicosapentaenoic acid, docosahexaenoic acid, arachydonic acid, oleic acid, eicosaenoic acid, docosaenoic acid or mixtures thereof are preferably used as fatty acids in the context of the present invention for re-esterification.
  • the extraction with supercritical fluids eliminated more than 95% of the content of squalene from the shark oil, producing a refined product with a content in said hydrocarbon of less than 4% by weight.
  • the transesterification was carried out between the ethyl ester of the conjugated linoleic acid (CLA) and the non-esterified alkoxyglycerols to produce alkoxyglycerols with conjugated linoleic acid derivatives. There was continuous nitrogen bubbling during the reaction to evaporate the ethanes caused by the reaction, which shifted the balance of the reaction towards the production of alkylglycerol diester with conjugated linoleic acid.
  • CLA conjugated linoleic acid
  • Supercritical fractionation is carried out with carbon dioxide in an installation with a filled column, in countercurrent conditions, to obtain a refined product with a 3% squalene content.
  • the extraction was carried out at a temperature of 6O 0 C and at a pressure of 180 bar, with a solvent-feed ratio of 45.
  • the ethereal phase was then dried with sodium sulfate and evaporated under nitrogen to obtain the unsaponifiable residue.
  • the unsaponifiable fraction (500 mg) and the CLA ethyl ester (818 mg) or the free fatty acid (CLA) was added to a 30 mL flask with stirring.
  • the lipase (10% by weight) was added, the flask was closed and placed in an orbital shaker (at 200 rpm) at 55 0 C. Samples were periodically obtained.
  • the reaction products were analyzed by preparing methyl esters of the free fatty acids, to that end 1 mL of 0.2 mole methanolic HCL at 250 ⁇ L was added and was left to stand for 4h at 6O 0 C.

Abstract

The invention relates to lipid compounds derived from alkoxyglycerols (or lipid ethers) with a beneficial activity for health, or which can be lipid carriers for administering bioactive ingredients. The invention further relates to the process of obtaining said modified alkoxyglycerols by means of extraction with supercritical fluids and subsequent enzymatic or chemical interesterification.

Description

MODIFIED ALKOXYGLYCEROLS
FIELD OF THE INVENTION
This invention relates to lipid compounds with biological activity and more specifically to lipid compounds derived from ether lipids (or alkoxyglycerols) with a beneficial activity for health, or which can be lipid carriers for the administration of bioactive ingredients. The invention further relates to the process for obtaining said modified alkoxyglycerols.
STATE OF THE ART
Shark liver oil is a natural source of certain alkoxyglycerols and other types of lipids such as triacylglycerols, squalene and cholesterol. Natural alkoxyglycerols or 1-O-alkyl-sn-glycerols are valuable compounds which can be isolated from shark liver oil. Different applications of such compounds naturally present in fish oils, and particularly in shark liver oils, have been described, such as the use of these 1 -O-alkyl-glycerols for preserving or improving sperm in pharmaceutical or veterinary uses (ES 2221057, W9800120). In this case, a 1 -O-alkyl-glycerol compound chosen from the group comprising 16:0 1 -alkyl-glycerol, 18:1 1 -alkyl-glycerol and 16.1 1 - alkylglycerol, which are ether lipids present in shark liver oil, is used.
Natural ether lipids (also known as alkoxyglycerols or alkyl glycerol ethers) have shown different beneficial effects for health. The oral administration of ether lipids reduces the negative effects of radiations received in radiation therapies. Prophylactic effects in certain tumors have also been observed. It has also been observed in experiments in both animals and humans that they are able to improve the immune response (Andreesen R., "Ether lipids in the therapy of cancer", Prog Biochem
Pharmacol 22: 118-131 , (1988), Palmblad, J, J Samuelsson, and J Brohult, "Interactions between alkylglycerols and human neutrophil granulocytes", Scand J Clin Lab Invest 50. 363-370 (1990)).
These ether lipids are found in bone marrow, breast milk, placenta and in shark liver oils in large amounts. In the human body, the ether lipids are mainly found in immune system cells and in breast milk. In some circumstances, the endogenous synthesis is reduced and in these cases, the administration of alkoxyglycerols is recommended.
To isolate natural alkoxyglycerols, it is necessary to eliminate other components of shark oil, such as squalene and fatty acids. Supercritical fluid technology, which has demonstrated very interesting properties, can be used to that end (Mukhopadhyay, M., Natural extracts using supercritical Carbon dioxide. CRC press. Boca Raton, Florida, 2000).
To produce fats and oils enriched with long chain polyunsaturated fatty acids and/or with conjugated linoleic acid, they can be enzymatically enriched.
Reactions catalyzed by esterification enzymes (Shimada, Y., A
Sugihara, H Nakano, T. Kuramoto, T. Nagao. M. Gemba, and Y. Tominaga, Purification of Docosahexaenoic Acid by Selective Esterification of Fatty Acids from Tuna Oil with Rhizopus delemar Lipase, J Am. Oil Chem Soc 74: 97-101 (1997)), hydrolysis (McNeil, G. P, R.G Ackman, and B. Kristinsson, Lipase-catalyzed enrichment of long-chain polyunsaturated fatty acids, J Am
Oil Chem Soc 73: 1403-1407(1996)) transestehfication (Breivik, H., G. G Haraldsson, and B Kristinsson, Preparation of Highly Purified Concentrates of Eicosapentaenoic Acid and Docosahexaenoic Acid, J Am Oil Chem Soc. 74, 1425-1429, 1997)), and acidolysis (Torres, C. F., F. Munir, L. P Lessard, and Charles G. Hill Jr., Lipase-Mediated Acidolysis of Thstearin with CLA in a
Packed-Bed Reactor: A Kinetic Study, J Am Oil Chem Soc. 79 655-661 , (2002)) have been used to obtain selective enrichments of natural substances with conjugated linoleic acid (CLA) and with polyunsaturated fatty acids.
One of the fatty acids that has attracted most the attention in the recent years is conjugated linoleic acid (CLA), which is formed by a mixture of positional and geometric isomers of octadecadienoic acid with conjugated double bonds. Nutritional studies based on animal models have demonstrated a wide variety of beneficial effects derived from ingesting CLA in the diet. These effects include their anticarcinogenic, antiatherogenic, antiobesity properties and their immune system enhancing effect (Whigham, L. D., M. E. Cook, R. L. Atkinson, "Conjugated Linoleic Acid. Implications for Human Health", Pharmacol Res. 42: 503-510, (2000), Pariza, M.W., Y. Park, M. E. Cook, "The Biologically Active Isomers of Conjugated Linoleic Acid", Prog. Lipid Res. 40: 283-298, (2001 )).
Since the middle of the 20th century, research has been carried out aimed at learning the effects of the polyunsaturated fatty acids or PUFA (Polyunsaturated Fatty Acids) in reducing serum cholesterol levels and in cardiovascular diseases. The most relevant works in this sense where those of Ahrens et al., 1954 (Ahrens E. H., D. H. Blankenhorn, TT. Tastas (1954), "Effect on human serum lipids of substituting plant for animal fat in the diet", Proc. Soc. Exp. Biol. Med 86, 872) and Keys et al., 1957 (Keys A, JT. Anderson, F. Grande (1957), "Serum cholesterol response to dietary fat", Lancet 1 , 787) which provided clear evidence regarding the importance of
PUFA in preventing cardiovascular diseases. Many studies have been conducted since then to that effect, most of which have confirmed the heart- healthy effects of omega-3. For example, in a clinical trial recently conducted by researchers at the Laboratory of Cardiovascular Nutrition of the Baker Medical Research Institute of Melbourne, the Department of Medicine of the
Medical Defense College of Tokyo, the CSIRO of the Division of Health Sciences and Nutrition of Adelaide (Australia) and of Vitamin Research of F Hoffmann-La Roche (Switzerland) published in the American Journal of Clinical Nutrition (Am J Clin Nutr 76 (2002) 326-330) of the American Society for Clinical Nutrition, it has been demonstrated that omega-3 fatty acids, especially long-chain omega-3 fatty acids, i.e. DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid), have effects on the maintenance of arterial elasticity and, accordingly, on the maintenance of normal blood pressure levels and the reduction of cardiovascular risk. The study consisted of administering DHA or EPA or placebo to patients with hypercholesterolemia for seven weeks. The researchers then determined elasticity of the arteries of the participants through ultrasounds. Those who received co-3 fatty acids showed a significant reduction in arterial sclerosis, whereas those who took placebo experienced no changes. Those who took EPA showed a 36% increase in arterial systemic resistance, a measure of elasticity of the main arteries, whereas those who took DHA showed a 27% increase.
Omega-3 fatty acids (EPA/DHA) improve the blood lipid profile because they increase elasticity, decrease LDL cholesterol, increase HDL cholesterol, reduce arterial triglycerides and are antithrombotic. Adeemia (Lόpez-Huertas-E; Baro, L.; Carrero, J J; Fonolla, J (2003) "n-3 fatty acids: health effects and opportunities to increase intake", Agro Food Industry hi tech 2003, 14(3). 18-21 ; Dewailly, E, Blanchet, C, Gingras S; Lemieux, S, Holub, B J (2002), "Cardiovascular disease risk factors and n-3 fatty acid status in the adult population of James Bay Cree", American Journal of Clinical Nutrition 2002; 76(1 ), 85-92).
In addition to the heart-healthy effects of omega-3, and as discussed in preceding paragraphs, these fatty acids have important effects on gene expressions and other biochemical body processes. Among other functions, omega-3 stands out due to its involvement in forming cell membranes. Most brain tissues are rich in omega-3 fatty acids. The current knowledge of these effects is included in an article by Donald B. Jump of the Department of Physiology, Biochemistry, and Molecular Biology of Michigan State University published in the Journal of Biological Chemistry of the American Society for Biochemistry and Molecular Biology (J Biol Chem 227 (2002) 8755-8758).
One way of assuring suitable intake of these fatty acids and of CLA is the production of lipids rich in these substances. Ether lipids can furthermore be suitable as lipid carriers to assure higher bioavailability and a better response of these substances. In addition, the combined administration of both, fatty acids and alkoxyglycerols, in a single molecule enhances the beneficial effect that both molecules have separately.
OBJECT OF THE INVENTION
A first object of the invention is represented by modified alkoxyglycerols of general formula CH2OR1-CHOR2-CH2OR3 where Ri, R2 and R3 independently represent a hydrogen; a saturated or unsaturated, linear or branched C2-C24 alkyl group, with or without double bonds, or a conjugated or unconjugated, saturated or unsaturated, linear or branched d- C24 acyl group with or without double bonds where at least one of Ri, R2 or
R3 represents an alkyl group.
A second object is the process for obtaining said modified alkoxyglycerols comprising three steps: supercritical fractionation of the natural alkoxyglycerols, saponification and re-esterification thereof.
Finally, another object of the invention is also the use of the modified alkoxyglycerols of the invention to prevent cardiovascular diseases, as an immunostimulant or as an anticarcinogen. Modified alkoxyglycerols are also useful as carrier agents for other active ingredients and bioactive substances, making them suitable to form part of food compositions in functional foods or as an additive in pharmaceutical compositions.
DESCRIPTION OF THE INVENTION
A first aspect of the present invention relates to modified alkoxyglycerols of general formula CH2OR1-CHOR2-CH2OR3 where Ri, R2 and R3 independently represent a hydrogen; a saturated or unsaturated, linear or branched C2-C24 alkyl group, with or without double bonds; or a conjugated or unconjugated, saturated or unsaturated, linear or branched d-
C24 acyl group with or without double bonds where at least one of Ri, R2 or R3 represents an alkyl group.
In a preferred embodiment of the invention the modified alkoxyglycerols Ri is a saturated or unsaturated, linear or branched C2-C24 alkyl; R2 is a hydroxyl group or an alkyl or acyl group, both being C2-C24 and
R3 is a hydroxyl group or an alkyl or acyl group, both being conjugated or unconjugated, saturated or unsaturated, linear or branched C2-C24 with or without double bonds where at least one of R2 or R3 represents an acyl group.
In a specific embodiment, the acyl group or groups come from saturated fatty acids with 1 -10 carbons.
Furthermore, in a preferred embodiment the acyl groups have one or more unsaturations. It is also preferred that these unsaturations are conjugated as, for example, in the case of conjugated linoleic acid (CLA) which, as an acyl substituent in the general formula, would be in the form of conjugated linoleyl radical. Modified alkoxyglycerols with long-chain polyunsaturated fatty acids (PUFA) of the omega-3 or omega-6 family, such as for example docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA), are also a preferred embodiment.
Finally, it is also preferred that the acyl group or groups in the modified alkoxyglycerols of the invention come from oleic acid, therefore representing an oleyl radical.
In addition, when R1 , R2 or R3 represent an alkyl group it is preferred that they have an even number of carbons.
The modified alkoxyglycerols of the invention (referred to as such hereinafter) are useful for preventing cardiovascular diseases and are also useful as stimulants of the immune system and anticarcinogenic. This activity comes from the beneficial effect provided by the fatty acids, whereby modifying the alkoxyglycerols, and by the ether lipids themselves. The modified alkoxyglycerols of the invention can be used in both the food and pharmaceutical industry for preparing functional products and drugs.
In fact, the alkoxyglycerols of the invention can be used as carrier agents of other bioactive compounds in food or pharmaceutical compositions. For example, the modified alkoxyglycerols of the invention can act as carrier agents of fatty acids of the omega-3, omega-6, omega-9 (oleic) and/or CLA family. A synergistic activity can thus be reached between the alkoxyglycerols of the invention and fatty acids carried by them.
Another aspect of the invention is the process for obtaining the modified alkoxyglycerols of the invention. This process comprises: a) countercurrent column supercritical fractionation of shark liver oil for obtaining a refined product rich in alkoxyglycerols and an extract rich in squalene, b) saponification of the refined product rich in alkoxyglycerols of a) for producing an unsaponifiable fraction rich in non-esterified alkoxyglycerols, and c) chemical or enzymatic re-esterification of the product of b) with aliphatic, saturated or unsaturated, conjugated or unconjugated, linear or branched chain C2-C24 monocarboxylic acids.
As a step prior to supercritical fractionation, the shark liver oil can be subjected to another optional supercritical extraction step to extract squalene and other lipid products present in the oil, such as cholesterol or triacylglycerols, to obtain a refined product rich in even purer alkoxyglycerols.
Supercritical fractionation and extraction process
Supercritical fractionation is carried out in a filled column to separate ether lipid components from edible oil samples containing them. Shark liver oil is particularly preferred due to its high content in alkoxyglycerols. The extraction is carried out with supercritical fluids, preferably carbon dioxide, on the alkoxyglycerols source (shark liver oil, or from a shark oil chemically or enzymatically modified or modified by saponification). The fractionation is carried out at an extraction pressure of between 90 and 320 bar, preferably 9-270 bar, and at a temperature of between 1 O0C and 9O0C. A refined product rich in alkoxyglycerols (less than 4% of squalene) and an extract rich in squalene are obtained after fractionation.
Saponification process The refined product rich in alkoxyglycerols obtained after supercritical fractionation is still unsuitable for esterification because it still has a high percentage of thacylglycerols that can cause unwanted esterification and transestehfication reactions in the presence of lipases. Therefore, saponification is carried out before this step to eliminate all the triacylglycerols and to obtain an unsaponifiable fraction highly rich in non- estehfied alkoxyglycerols.
Saponification is carried out by means of treating the refined product rich in alkoxyglycerols with soda or potash or with an alkali in a basic medium. An unsaponifiable residue is obtained from this reaction, which product will be used in the transesterification step.
Enzymatic or chemical transestehfication process
Esterification of the alkoxyglycerols is preferably carried out enzymatically with a lipase in the presence of C2-C24 fatty acids or with esters thereof, with which the alkoxyglycerols are to be estehfied. The reaction catalyzed by the lipase takes place in two consecutive steps. An alkylglycerol monoester is formed during the first acylation and the alkylglycerol diester is formed during a second acylation. In other words, modified alkoxyglycerols, different from natural alkoxyglycerols, are thus obtained.
Conjugated linoleic acid (CLA), eicosapentaenoic acid, docosahexaenoic acid, arachydonic acid, oleic acid, eicosaenoic acid, docosaenoic acid or mixtures thereof are preferably used as fatty acids in the context of the present invention for re-esterification.
EXAMPLE: Process for producing modified alkoxyglycerols with a high content of substituents of fatty conjugated linoleic acid (CLA) from shark liver oil An extraction was carried out with supercritical fluids preferably of shark liver oil, or of a shark oil modified chemically or enzymatically or by saponification, followed by saponification of the refined product rich in alkoxyglycerols and a subsequent reaction of the unsaponifiable fraction with
CLA or CLA ethyl ester.
The extraction with supercritical fluids eliminated more than 95% of the content of squalene from the shark oil, producing a refined product with a content in said hydrocarbon of less than 4% by weight.
This refined product was still unsuitable to produce modified alkoxyglycerols because it contained a high percentage of triacylglycerols, which could cause unwanted esterification and transestehfication reactions in the presence of lipases. Therefore, a saponification reaction was carried out to effectively eliminate all the triacylglycerols and to obtain an unsaponifiable fraction highly rich in non-esterified alkoxyglycerols.
The transesterification was carried out between the ethyl ester of the conjugated linoleic acid (CLA) and the non-esterified alkoxyglycerols to produce alkoxyglycerols with conjugated linoleic acid derivatives. There was continuous nitrogen bubbling during the reaction to evaporate the ethanes caused by the reaction, which shifted the balance of the reaction towards the production of alkylglycerol diester with conjugated linoleic acid.
Extraction with supercritical fluids
Supercritical fractionation is carried out with carbon dioxide in an installation with a filled column, in countercurrent conditions, to obtain a refined product with a 3% squalene content. The extraction was carried out at a temperature of 6O0C and at a pressure of 180 bar, with a solvent-feed ratio of 45.
Saponification
5 grams of refined product were mixed with 16 mL of 3.7 N potassium hydroxide solution in ethanol (water 50:50 v:v with 0.15% of EDTA). The mixture was heated at 6O0C for one hour at 300 rpm. The reaction was stopped by adding 4 mL of water and the unsaponifiable product was extracted from the aqueous phase with 3 portions of 20 mL of diethyl ether.
The ethereal phase was then dried with sodium sulfate and evaporated under nitrogen to obtain the unsaponifiable residue.
Transesterification reaction
The unsaponifiable fraction (500 mg) and the CLA ethyl ester (818 mg) or the free fatty acid (CLA) was added to a 30 mL flask with stirring. The lipase (10% by weight) was added, the flask was closed and placed in an orbital shaker (at 200 rpm) at 550C. Samples were periodically obtained. The reaction products were analyzed by preparing methyl esters of the free fatty acids, to that end 1 mL of 0.2 mole methanolic HCL at 250 μL was added and was left to stand for 4h at 6O0C.
After adding 200 μL of water, the mixture was extracted, dried and centhfuged to obtain the compounds which were analyzed by gas chromatography.

Claims

1. Modified alkoxyglycerols of general formula CH2ORrCHOR2- CH2OR3 where Ri, R2 and R3 independently represent between a hydrogen group; a saturated or unsaturated, linear or branched C2-C24 alkyl group, with or without double bonds, or a conjugated or unconjugated, saturated or unsaturated, linear or branched Ci-C24 acyl group with or without double bonds, characterized in that at least one of Ri, R2 or R3 represents an alkyl group.
2. Modified alkoxyglycerols according to claim 1 , where Ri is a saturated or unsaturated, linear or branched C2-C24 alkyl; R2 is a C2-C24 hydrogen or alkyl group or CrC24 acyl group, and R3 is a C2-C24 hydrogen or alkyl group or CrC24 acyl group, both being conjugated or unconjugated, saturated or unsaturated, linear or branched with or without double bonds, characterized in that at least one of R2 or R3 represents an acyl group.
3. Modified alkoxyglycerols according to claim 1 , characterized in that at least one of Ri, R2 and R3 represents a saturated acyl group of 1 -10 carbons.
4. Modified alkoxyglycerols according to claim 1 , characterized in that at least one of Ri, R2 and R3 is an acyl group with one or more unsaturations.
5. Modified alkoxyglycerols according to claim 1 , characterized in that at least one of Ri, R2 and R3 represents a conjugated linoleyl group.
6. Modified alkoxyglycerols according to claim 1 , characterized in that at least one of Ri, R2 and R3 represents an acyl group corresponding to that of a long-chain polyunsaturated acid (PUFA) of the omega-3 or omega-6 family.
7. Modified alkoxyglycerols according to claim 1 , characterized in that at least one of Ri, R2 and R3 represents an oleyl group.
8. Modified alkoxyglycerols according to claim 1 , characterized in that at least one of Ri, R2 and R3 represents an alkyl group with an even number of carbon atoms.
9. Modified alkoxyglycerols according to claim 1 for their use in preventing cardiovascular diseases.
10. Modified alkoxyglycerols according to claim 1 for their use as an immune system stimulant.
11. Modified alkoxyglycerols according to claim 1 for their use as an anticarcinogen.
12. A process for preparing modified alkoxyglycerols according to claim 1 , characterized in that it comprises:
a) supercritical fractionation in countercurrent column of shark liver oil for obtaining a refined product rich in alkoxyglycerols and an extract rich in squalene,
b) saponification of the refined product rich in alkoxyglycerols of a) for producing an unsaponifiable fraction rich in non-estehfied alkoxyglycerols, and
c) chemical or enzymatic re-esterification of the product of b) with conjugated or unconjugated, aliphatic, saturated or unsaturated, linear or branched chain C2-C24 monocarboxylic acids.
13. A process according to claim 12, characterized in that the supercritical fluid used for fractionation is carbon dioxide.
14. A process according to claim 12, characterized in that the extraction pressure in the column is between 90 and 320, preferably 90 and
270 bar, and the extraction temperature is between 1 O0C and 9O0C.
15. A process according to claim 12, characterized in that saponification is carried out with soda, with potash or in a basic medium with an alkali.
16. A process according to claim 12, characterized in that re- estehfication is an enzymatic process and is carried out with lipase.
17. A process according to claim 12, characterized in that the C2-C24 monocarboxylic acids used in re-estehfication are the conjugated linoleic acid (CLA), eicosapentaenoic acid, docosahexaenoic acid, arachydonic acid, oleic acid, eicosaenoic acid, docosaenoic acid or mixtures thereof.
18. Modified alkoxyglycerols that can be obtained by the process of claims 12 to 17.
19. The use of modified alkoxyglycerols according to any of claims 1 - 11 for the manufacture of food or pharmaceutical compositions to prevent cardiovascular diseases.
20. The use of modified alkoxyglycerols according to any of claims 1 - 11 for the manufacture of food or pharmaceutical compositions to stimulate the immune system.
21. The use of modified alkoxyglycerols according to any of claims 1 - 11 for the manufacture of food or pharmaceutical compositions with anticarcinogenic activity.
22. The use of modified alkoxyglycerols according to claims 1 -11 as carrier agents of other or others bioactive compounds in food or pharmaceutical compositions.
23. The use according to claim 22 where bioactive compounds synergistically act with the alkoxyglycerols simultaneously acting as carrier agents.
24. The use according to claim 23 where the bioactive compounds are fatty acids of the omega 3 and/or CLA family.
EP07819930.4A 2006-09-29 2007-08-10 Modified alkoxyglycerols Not-in-force EP2079824B1 (en)

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PCT/EP2007/058315 WO2008037538A2 (en) 2006-09-29 2007-08-10 Modified alkoxyglycerols

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AU288098B1 (en) * 1963-08-30 1966-02-24 Astra Nutritionab The production of products of nutritional value for fish or animals containing glycerol ethers
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